The call for a new toxicology is mainly a call for cellular approaches and their computational integration. This article reflects on cell models, which are necessary to facilitate the transition. A mechanistic perspective has prompted the characterization of toxicity pathways and toxicity networks in order to develop robust cell-based assays for toxicity testing. Differing use scenarios for cell systems require higher degrees of sophistication, e.g., human-on-a-chip approaches are based on complex organotypic cultures to approximate the repertoire of human physiological reactions and high-throughput tests require simplicity and robustness. The new paradigm emerging under the branding of Toxicology for the 21st Century needs complex models for pathway of toxicity identification and simpler assays for testing the perturbation of any given pathway. With increasing knowledge about underlying mechanisms, the needs for complexity and test specificity will change. Selective cell-based assays are desirable, especially for the detection of novel toxicants and biothreats. Examples from endocrine disruption, pyrogenicity, and especially shellfish toxin testing are used to illustrate such developments.

Adverse hypertrophic scars can form after healing of full-thickness skin wounds. Currently, reliable animal and in vitro models to identify and test novel scar reducing therapeutics are scarce. Here we describe the development and validation of a tissue-engineered human hypertrophic scar (HTscar) model based on reconstructed epidermis on a dermal matrix containing adipose derived mesenchymal stem cells (ASC). Although obtained from normal, healthy skin, ASC, in contrast to dermal mesenchymal cells, were found to facilitate HTscar formation. Quantifiable HTscar parameters were identified: contraction; thickness of dermis, collagen-1 secretion, epidermal outgrowth, epidermal thickness, and cytokine secretion (IL-6, CXCL8). The model was validated with therapeutics currently used for treating scars (5-fluorouracil, triamcinolon) and a therapeutic known to be unsuccessful in scar reduction (1,25-dihydroxyvitamin-D₃). Furthermore, it was shown that atorvastatin, but not retinoic-acid, may provide a suitable alternative for scar treatment. Each therapeutic selectively affected a different combination of parameters, suggesting combined therapy may be most beneficial. This animal-free hypertrophic scar model may provide an alternative model for mechanistic studies as well as a novel in vitro means to test anti-scar therapeutics, thereby reducing the use of animals.

The role that in vitro systems can play in toxicological risk assessment is determined by the appropriateness of the chosen methods, with respect to the way in which in vitro data can be extrapolated to the in vivo situation. This report presents the results of a workshop aimed at better defining the use of in vitro-derived biomarkers of toxicity (BoT) and determining the place these data can have in human risk assessment. As a result, a conceptual framework is presented for the incorporation of in vitro-derived toxicity data into the risk assessment process. The selection of BoT takes into account that they need to distinguish adverse and adaptive changes in cells. The framework defines the place of in vitro systems in the context of data on exposure, structural and physico-chemical properties, and toxicodynamic and biokinetic modeling. It outlines the determination of a proper point-of-departure (PoD) for in vitro-in vivo extrapolation, allowing implementation in risk assessment procedures. A BoT will need to take into account both the dynamics and the kinetics of the compound in the in vitro systems. For the implementation of the proposed framework it will be necessary to collect and collate data from existing literature and new in vitro test systems, as well as to categorize biomarkers of toxicity and their relation to pathways-of-toxicity. Moreover, data selection and integration need to be driven by their usefulness in a quantitative in vitro-in vivo extrapolation (QIVIVE).

Several alternative methods to replace animal experiments have been accepted by legal bodies. An even larger number of tests are under development or already in use for non-regulatory applications or for the generation of information stored in proprietary knowledge bases. The next step for the use of the different in vitro methods is their combination into integrated testing strategies (ITS) to get closer to the overall goal of predictive “in vitro-based risk evaluation processes.” We introduce here a conceptual framework as the basis for future ITS and their use for risk evaluation without animal experiments. The framework allows incorporation of both individual tests and already integrated approaches. Illustrative examples for elements to be incorporated are drawn from the session “Innovative technologies” at the 8^th World Congress on Alternatives and Animal Use in the Life Sciences, held in Montreal, 2011. For instance, LUHMES cells (conditionally immortalized human neurons) were presented as an example for a 2D cell system. The novel 3D platform developed by InSphero was chosen as an example for the design and use of scaffold-free, organotypic microtissues. The identification of critical pathways of toxicity (PoT) may be facilitated by approaches exemplified by the MatTek 3D model for human epithelial tissues with engineered toxicological reporter functions. The important role of in silico methods and of modeling based on various pre-existing data is demonstrated by Altamira’s comprehensive approach to predicting a molecule’s potential for skin irritancy. A final example demonstrates how natural variation in human genetics may be overcome using data analytic (pattern recognition) techniques borrowed from computer science and statistics. The overall hazard and risk assessment strategy integrating these different examples has been compiled in a graphical work flow.

The introduction of technical surgical innovations in clinical medicine is preceded by preclinical evaluation of prototypes. Surgical aspects such as energy dependent tissue response and tissue sealing to reduce bleeding are usually investigated in animal experiments. Extra-corporal organ models can provide the required experimental information without harming animals and thus reduce or even replace in vivo experiments. Here we describe the ex vivo ox-foot-model, which can be used for surgical investigations and for training purposes.
In the ox-foot-model the vein remains in its anatomical bed under reproducible experimental conditions, i.e., blood perfusion, blood pressure, and temperature. Innovative endoluminal surgical procedures using laser light and radio frequency for varicosis treatment were tested. Treatment parameters were investigated systematically in a large number of samples. A standardized preclinical testing procedure could be established and optimized on the basis of acute macroscopic and histological findings. Further, optical coherence tomography could be evaluated as a time-saving diagnostic tool. The ox-foot-model is suitable for training surgical techniques relevant for the treatment of varicosis veins.
It is a cost-effective alternative to conventional in vivo experiments, providing standardized experimental conditions and reproducible experimental results while respecting the Principles of Humane Experimental Techniques: Reduction, Refinement, and Replacement of animal experiments.